Antimicrobial peptides (AMPs) are intrinsic host defence molecules which are probably produced by all multicellular plants and animals (see “Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389-395 (2002)”). They comprise the first line of innate immune system to rapidly eliminate invading pathogens in the early stage of infection and can also promote systemic adaptive immune response (see “Rohrl, J., Huber, B., Koehl, G. E., Geissler, E. K. & Hehlgans, T. Mouse beta-defensin 14 (Defb14) promotes tumor growth by inducing angiogenesis in a CCR6-dependent manner. J Immunol 188, 4931-4939 (2012)” and “Yang, D., et al. Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286, 525-528 (1999)”). Most AMPs are amphipathic and cationic molecules, which confer the binding ability to the microbe membranes that are generally negatively charged. Many hundreds of AMPs have been identified and classified according to their structural features and/or amino acid compositions. Two families of AMPs in vertebrates, cathelicidins and defensins, are small molecules mainly produced by leucocytes and epithelia cells (see “Lehrer, R. I. Primate defensins. Nat Rev Microbiol 2, 727-738 (2004)” and “Selsted, M. E. & Ouellette, A. J. Mammalian defensins in the antimicrobial immune response. Nat Immunol 6, 551-557 (2005)”). The precursors of cathelicidins contain a conserved amino-terminal “cathelin” domain (about 100-residue-long). Processed cathelicidin peptides range in length from 12 to 80 amino acid residues, with or without α-helical, β-sheet or other types of tertiary structures (see “Lehrer, R. I. & Ganz, T. Cathelicidins: a family of endogenous antimicrobial peptides. Curr Opin Hematol 9, 18-22 (2002)” and “Zanetti, M. Cathelicidins, multifunctional peptides of the innate immunity. J Leukoc Biol 75, 39-48 (2004)”). Defensins are small (2-6 kD) cysteine-rich AMPs which mainly form β-sheet structures stabilized by three (rarely four) conserved intramolecular disulphide bridges. Three subfamilies of defensins are further classified as α-, β- and θ-defensins in vertebrates according to their disulfide patterns (see “Lehrer, R. I. Primate defensins. Nat Rev Microbiol 2, 727-738 (2004)”). These peptides generally have a broader range of non-specific activity against infections of microorganisms, including gram-positive and gram-negative bacteria, fungi and viruses. The diverse action modes of AMPs against bacteria include disrupting membrane integrity (see “Shai, Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta 1462, 55-70 (1999)” and “Yang, L., Weiss, T. M., Lehrer, R. I. & Huang, H. W. Crystallization of antimicrobial pores in membranes: magainin and protegrin. Biophys J 79, 2002-2009 (2000)”), impairing nucleus and protein synthesis, inhibiting chaperone-assisted protein fold, interrupting cell-wall biosynthesis pathway and targeting membrane biogenesis (see “Brogden, K. A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3, 238-250 (2005)”, “Hale, J. D. & Hancock, R. E. Alternative mechanisms of action of cationic antimicrobial peptides on bacteria. Expert Rev Anti Infect Ther 5, 951-959 (2007)” and “Srinivas, N., et al. Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa. Science 327, 1010-1013 (2010)”). Thus far, however, the antiviral mechanism of AMPs is still largely unknown.
Defensins have been shown to possess many properties, including antibacterial (see “Nizet, V., et al. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 414, 454-457 (2001)” and “Mygind, P. H., et al. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature 437, 975-980 (2005)”), antiviruses (see “Gong, T., et al. Recombinant mouse beta-defensin 2 inhibits infection by influenza A virus by blocking its entry. Arch Virol 155, 491-498 (2010)” and “Leikina, E., et al. Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins. Nat Immunol 6, 995-1001 (2005)”) and antifugi (see “Krishnakumari, V., Rangaraj, N. & Nagaraj, R. Antifungal activities of human beta-defensins HBD-1 to HBD-3 and their C-terminal analogs Phd1 to Phd3. Antimicrob Agents Chemother 53, 256-260 (2009)” and “Jiang, Y., et al. Antifungal activity of recombinant mouse beta-defensin 3. Lett Appl Microbiol 50, 468-473 (2010)”).
Native defensins are produced by innate and adaptive immune systems in response to infections, such as viral infection (see “Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389-395 (2002)”). In mice, gene expression of murine β-defensin-3 and β-defensin-4 is induced in upper respiratory tract by influenza virus infection (see “Chong, K. T., Thangavel, R. R. & Tang, X. Enhanced expression of murine beta-defensins (MBD-1, -2, -3, and -4) in upper and lower airway mucosa of influenza virus infected mice. Virology 380, 136-143 (2008)”). HIV-1 Tat can induce human β-defensin-2 expression in human B cells (see “Ju, S. M., et al. Extracellular HIV-1 Tat induces human beta-defensin-2 production via NF-kappaB/AP-1 dependent pathways in human B cells. Mol Cells 33, 335-341 (2012)”).
The induced defensins play important roles in the protection against invading microbes in the early stage of infection (see “Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389-395 (2002)”). Human α-defensin-1 has been demonstrated to inhibit influenza virus replication, which may be due to attenuation of protein kinase C activation (see “Salvatore, M., et al. alpha-Defensin inhibits influenza virus replication by cell-mediated mechanism(s). J Infect Dis 196, 835-843 (2007)”). Mouse β-denfensin-2 has been demonstrated to inhibit viral infection by blocking viral entry into target cells (see “Gong, T., et al. Recombinant mouse beta-defensin 2 inhibits infection by influenza A virus by blocking its entry. Arch Virol 155, 491-498 (2010)”). A θ-defensin, retrocylin 2 (RC2), was illustrated to inhibit virus-cell membrane fusion by crosslinking membrane glycoproteins (see “Leikina, E., et al. Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins. Nat Immunol 6, 995-1001 (2005)”). Effectors of adaptive immune system, such as antibodies and T lymphocytes, are highly pathogen specific. In contrast, effectors of innate immune system, such as defensins, generally have broader spectrum activity against microorganisms. The unique properties of defensins make them attractive candidates for development of broader spectrum antiviral drugs with reduced opportunity of drug-resistance. For antiviral strategies, inhibition of viral entry, viral RNA release, virus replications and release may be selected as the targets for development of antiviral drugs.
Specific antivirals against common respiratory virus families, such as orthomyxoviridae, paramyxoviridae and coronaviridae causing emerging infections, are either not available or prone to develop drug-resistance due to the rapid mutation of these viral genes (see “Cheng, V. C., S. K. Lau, P. C. Woo, and K. Y. Yuen. 2007. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin Microbiol Rev 20:660-694”, “Cheng, V. C., K. K. To, H. Tse, I. F. Hung, and K. Y. Yuen. 2012. Two years after pandemic influenza A/2009/H1N1: what have we learned? Clin Microbiol Rev 25:223-263”, and “Wong, S. S., and K. Y. Yuen. 2008. Antiviral therapy for respiratory tract infections. Respirology 13:950-971”). Although many defensins from mice or humans have been found to have antiviral activity in vitro and in vivo (see “Jiang, Y., Y. Wang, Y. Kuang, B. Wang, W. Li, T. Gong, Z. Jiang, D. Yang, and M. Li. 2009. Expression of mouse beta-defensin-3 in MDCK cells and its anti-influenza-virus activity. Arch Virol 154:639-647”, “Quinones-Mateu, M. E., M. M. Lederman, Z. Feng, B. Chakraborty, J. Weber, H. R. Rangel, M. L. Marotta, M. Mirza, B. Jiang, P. Kiser, K. Medvik, S. F. Sieg, and A. Weinberg. 2003. Human epithelial beta-defensins 2 and 3 inhibit HIV-1 replication. Aids 17:F39-48”, and “Sun, L., C. M. Finnegan, T. Kish-Catalone, R. Blumenthal, P. Garzino-Demo, G. M. La Terra Maggiore, S. Berrone, C. Kleinman, Z. Wu, S. Abdelwahab, W. Lu, and A. Garzino-Demo. 2005. Human beta-defensins suppress human immunodeficiency virus infection: potential role in mucosal protection. J Virol 79:14318-14329”), the development of defensins as therapeutics has been hindered by several factors, such as suboptimal efficacy, side effects and the lack of cost-effective means of commercial-scale production.
Thus, a safe, potent and broad-spectrum antiviral is urgently needed to combat emerging viral respiratory diseases.